Although the progression of cancer arrives at a common end point of organ failure, cachexia and death, common pathways are rare in cancer. Over the past decade one common pathway consistently revealed by MR spectroscopic studies is the elevation of phosphocholine and total choline in cancer cells and solid tumors. Data obtained by us demonstrate that phosphocholine in cancer cells is related to malignant transformation, endothelial cancer cell interaction, invasion and metatasis. Therefore the elevation of phosphocholine presents a unique target to exploit for therapy. In this proposal we intend to develop molecular biology strategies to target choline kinase in oxygenated as well as hypoxic cells using small interfering RNA (siRNA) to inhibit the transcription of choline kinase. We have chosen choline kinase as a target since it is the enzyme which converts choline to phosphocholine. Choline kinase inhibition will be induced in normoxic cells using a mifepristone inducible system or in hypoxic cells using the hypoxia response element to drive the formation of the siRNA. In vivo, the hypoxia inducible system will also be combined with anti-angiogenic treatment which will create hypoxia over a larger region of the tumor. The effects of choline kinase inhibition with or without anti-angiogenic therapy on invasion, metabolism and vascularization will be determined with combined MRI and MRSI. Optical imaging will be used to detect hypoxia, and histology to detect metastasis. Microarray analyses will be performed characterize molecular alterations. These studies will be performed with three human breast cancer cell lines and their transgenic counterparts, studied as cells and as solid tumors in vivo. Cells in hypoxic environments in solid tumors are the most resistant to radiation and chemotherapy, and are most likely to lead to recurrence of the disease. Our cancer invasion studies have shown that the presence of endothelial cells in the proximity of cancer cells under hypoxic conditions confers an advantage in invasion of the cancer cells. Approaches to enhance the action of anti-angiogenic therapy, by exploiting the hypoxia induced by anti-angiogenic therapy to further target a key pathway in cancer cells would minimize the risk of increased invasion and act synergistically with anti-angiogenic therapy in killing cancer cells.